Bottom Line:
We determined the protective efficacy of one subclass - Sj-TSP-2e.Following the alignment of 211 cDNAs, we identified 7 clusters encoding S. japonicum TSP-2 (Sj-TSP-2) based on sequence variation in the large extracellular loop (LEL) region with differing frequency of transcription in male and female worms.We expressed in E. coli the LEL region of one of the clusters which exhibited a high frequency of transcription in female worms, and showed the purified recombinant protein (Sj-TSP-2e) was recognised by 43.1% of sera obtained from confirmed schistosomiasis japonica patients.

Background: Schistosoma mansoni tetraspanin 2 (Sm-TSP-2) has been shown to be strongly recognized by IgG1 and IgG3 antibodies from individuals putatively resistant to schistosome infection, but not chronically infected people, and to induce high levels of protection against challenge infection in the murine model of schistosomiasis. Amplification by PCR of homologous sequences from male and female S. japonicum worms showed the presence of 7 different clusters or subclasses of S. japonicum TSP-2. We determined the protective efficacy of one subclass - Sj-TSP-2e.

Methodology/principal findings: Following the alignment of 211 cDNAs, we identified 7 clusters encoding S. japonicum TSP-2 (Sj-TSP-2) based on sequence variation in the large extracellular loop (LEL) region with differing frequency of transcription in male and female worms. Quantitative PCR analysis revealed elevated expression of Sj-TSP-2 in adult worms compared with other life cycle stages. We expressed in E. coli the LEL region of one of the clusters which exhibited a high frequency of transcription in female worms, and showed the purified recombinant protein (Sj-TSP-2e) was recognised by 43.1% of sera obtained from confirmed schistosomiasis japonica patients. Vaccination of mice with the recombinant protein induced high levels of IgG1 and IgG2 antibodies, but no consistent protective efficacy against challenge infection was elicited in three independent trials.

Conclusions/significance: The highly polymorphic nature of the Sj-TSP-2 gene at the transcriptional level may limit the value of Sj-TSP-2 as a target for future S. japonicum vaccine development.

Mentions:
BLAST analysis showed that a number of S. japonicum sequences deposited in the GenBank and other databases were homologous to Sm-TSP-2. The Sj-TSP-2 sequences at both the N and C terminal ends are nearly identical, which allowed us to design primers to amplify the full-length ORF of the cDNAs. We sequenced 108 clones from male and 103 from female adult worms (Table 1). The ORFs of the sequences were identical in size, encoding a precursor protein of 215 amino acids. Protein sequence analysis showed that all the proteins have a similar structure containing four transmembrane domains and two loops (Figures S1 and S2). Whereas the N and C termini are highly conserved, the large loop region, which was used in the Sm-TSP-2 vaccine studies by Tran et al [5], is highly variable. The variation results in 7 clusters and Fig. 1 and Table 1 show representatives of each of the clusters. We compared the sequences with a previous study [8] and showed that 5 of the clusters were identical to those earlier reported as subclasses Sj-TSP-2a, c, d, e and f (Table 1). We identified two additional clusters, Sj-TSP-2h and Sj-TSP-2i (Table 1), but were unable to amplify any of the Sj-TSP-2b or Sj-TSP-2g sequences described previously [8].

Mentions:
BLAST analysis showed that a number of S. japonicum sequences deposited in the GenBank and other databases were homologous to Sm-TSP-2. The Sj-TSP-2 sequences at both the N and C terminal ends are nearly identical, which allowed us to design primers to amplify the full-length ORF of the cDNAs. We sequenced 108 clones from male and 103 from female adult worms (Table 1). The ORFs of the sequences were identical in size, encoding a precursor protein of 215 amino acids. Protein sequence analysis showed that all the proteins have a similar structure containing four transmembrane domains and two loops (Figures S1 and S2). Whereas the N and C termini are highly conserved, the large loop region, which was used in the Sm-TSP-2 vaccine studies by Tran et al [5], is highly variable. The variation results in 7 clusters and Fig. 1 and Table 1 show representatives of each of the clusters. We compared the sequences with a previous study [8] and showed that 5 of the clusters were identical to those earlier reported as subclasses Sj-TSP-2a, c, d, e and f (Table 1). We identified two additional clusters, Sj-TSP-2h and Sj-TSP-2i (Table 1), but were unable to amplify any of the Sj-TSP-2b or Sj-TSP-2g sequences described previously [8].

Bottom Line:
We determined the protective efficacy of one subclass - Sj-TSP-2e.Following the alignment of 211 cDNAs, we identified 7 clusters encoding S. japonicum TSP-2 (Sj-TSP-2) based on sequence variation in the large extracellular loop (LEL) region with differing frequency of transcription in male and female worms.We expressed in E. coli the LEL region of one of the clusters which exhibited a high frequency of transcription in female worms, and showed the purified recombinant protein (Sj-TSP-2e) was recognised by 43.1% of sera obtained from confirmed schistosomiasis japonica patients.

Background: Schistosoma mansoni tetraspanin 2 (Sm-TSP-2) has been shown to be strongly recognized by IgG1 and IgG3 antibodies from individuals putatively resistant to schistosome infection, but not chronically infected people, and to induce high levels of protection against challenge infection in the murine model of schistosomiasis. Amplification by PCR of homologous sequences from male and female S. japonicum worms showed the presence of 7 different clusters or subclasses of S. japonicum TSP-2. We determined the protective efficacy of one subclass - Sj-TSP-2e.

Methodology/principal findings: Following the alignment of 211 cDNAs, we identified 7 clusters encoding S. japonicum TSP-2 (Sj-TSP-2) based on sequence variation in the large extracellular loop (LEL) region with differing frequency of transcription in male and female worms. Quantitative PCR analysis revealed elevated expression of Sj-TSP-2 in adult worms compared with other life cycle stages. We expressed in E. coli the LEL region of one of the clusters which exhibited a high frequency of transcription in female worms, and showed the purified recombinant protein (Sj-TSP-2e) was recognised by 43.1% of sera obtained from confirmed schistosomiasis japonica patients. Vaccination of mice with the recombinant protein induced high levels of IgG1 and IgG2 antibodies, but no consistent protective efficacy against challenge infection was elicited in three independent trials.

Conclusions/significance: The highly polymorphic nature of the Sj-TSP-2 gene at the transcriptional level may limit the value of Sj-TSP-2 as a target for future S. japonicum vaccine development.